[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-548] Using Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates for Transient Analysis and Engineering of Bacterial Metabolic Pathways

○Yuka Kikuyama¹, Yugo Kawabuchi¹, Shunichi Kobayashi¹, Koji Sode², Ryutaro Asano¹, Tetsushi Mori¹ (1. Tokyo University of Agriculture and Technology (Japan), 2. University of North Carolina at Chapel Hill (USA))

Keywords:

Cell-penetrating peptides,Cyanobacteria,Metabolic pathway elucidation

Developing bacterial mutant strains is crucial for producing valuable natural compounds and understanding intracellular biochemistry. Conventional genetic engineering approaches for bacterial metabolic pathway manipulation face significant limitations including metabolic burden, irreversibility, dependency on host cellular machinery, and labor-intensive procedures. Cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs), recognized for their utility as antibacterial tools and in protein function elucidation, offer a promising alternative. Since CPP-PNA applications in metabolic engineering remain largely unexplored, we developed and validated a CPP-PNA platform as a rapid, reversible, non-genetic approach for assessing enzyme roles within metabolic pathways. Using the model cyanobacterium Synechocystis sp. PCC 6803, CPP-PNA constructs targeting essential housekeeping genes were designed and evaluated through growth inhibition assays. High compatibility and dose-dependent permeation efficiency was observed when amphipathic CPP (KFF)₃K was employed, achieving clear cell growth inhibition at 10 μM and above, demonstrating effective cellular permeation and translation inhibition within 24-48 hours. We selected D-lactate dehydrogenase (Ddh), a putative pyruvate metabolism regulator, as our target enzyme. A CPP-PNA probe specific to Ddh mRNA was synthesized and introduced into cultures. Specific targeting using CPP-Syn6803ddh conjugates achieved near-complete protein translation knockdown within 24 hours, as confirmed by Western blot analysis, reducing protein levels and causing significant pyruvate accumulation. Metabolomics analysis using LC-MS revealed that CPP-PNA treatment produced metabolic effects comparable to stable genetic knockout strains, with both approaches showing significant 2.5-fold pyruvate accumulation compared to wild-type controls. We observed compensatory activation of the glyoxalase pathway at 48 hours post-treatment, resulting in 3-fold increased D-lactate production, presumably through methylglyoxal detoxification. RT-qPCR analysis confirmed 2-3-fold upregulation of glyII expression in both CPP-PNA treated and knockout strains, while dual CPP-PNA inhibition targeting both Ddh and glyoxalase pathways effectively suppressed D-lactate accumulation. This study establishes CPP-PNAs as efficient tools for rapid metabolic pathway investigation. The approach produces results comparable to conventional genetic knockouts while offering dose-dependent control, time-resolved protein expression modulation, and avoiding permanent genomic alterations. Our findings reveal unexpected metabolic complexity in Synechocystis sp. PCC6803 D-lactate synthesis and demonstrate CPP-PNA utility for uncovering compensatory pathway activation. By enabling streamlined candidate enzyme identification for strain engineering, this platform represents a valuable addition to bacterial genetic engineering toolkits, addressing critical limitations of conventional approaches while enhancing understanding of bacterial metabolic networks.